Reduced tumorigenicity of rat glioma cells in the brain when mediated by hygromycin phosphotransferase

2001 ◽  
Vol 94 (4) ◽  
pp. 596-604 ◽  
Author(s):  
Adília Hormigo ◽  
David R. Friedlander ◽  
Perry A. Brittis ◽  
David Zagzag ◽  
Martin Grumet
Keyword(s):  
Cell Proliferation ◽  
Rat Brain ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells ◽  
Content Type ◽  
Hygromycin Phosphotransferase ◽  
The Brain ◽  
Fine Print

Object. A variant of C6 glioma cells, C6R-G/H cells express hygromycin phosphotransferase (HPT) and appear to have reduced tumorigenicity in the embryonic brain. The goal of this study was to investigate their reduced capacity to generate tumors in the adult rat brain. Methods. Cell lines were implanted into rat brains and tumorigenesis was evaluated. After 3 weeks, all rats with C6 cells showed signs of neurological disease, whereas rats with C6R-G/H cells did not and were either killed then or allowed to survive until later. Histological studies were performed to analyze tumor size, malignancy, angiogenesis, and cell proliferation. Cells isolated from rat brain tumors were analyzed for mutation to HPT by testing their sensitivity to hygromycin. Conclusions. The results indicate that HPT suppresses tumor formation. Three weeks after implantation, only 44% of animals implanted with C6R-G/H cells developed tumors, whereas all animals that received C6 glioma cells developed high-grade gliomas. The C6R-G/H cells filled a 20-fold smaller maximal cross-sectional area than the C6 cells, and exhibited less malignant characteristics, including reduced angiogenesis, mitosis, and cell proliferation. Similar results were obtained in the brain of nude rats, indicating that the immune system did not play a significant role in suppressing tumor growth. The combination of green fluorescent protein (GFP) and HPT was more effective in suppressing tumorigenesis than either plasmid by itself, indicating that the GFP may protect against inactivation of the HPT. Interestingly, hygromycin resistance was lost in tumor cells that were recovered from a group of animals in which C6R-G/H cells formed tumors, confirming the correlation of HPT with reduced tumorigenicity.

Inhibitory effect of antisense epidermal growth factor receptor RNA on the proliferation of rat C6 glioma cells in vitro and in vivo

2000 ◽  
Vol 92 (1) ◽  
pp. 132-139 ◽  
Author(s):  
Peiyu Pu ◽  
Xuwen Liu ◽  
Aixue Liu ◽  
Jianling Cui ◽  
Yunting Zhang
Keyword(s):  
Survival Time ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells ◽  
Content Type ◽  
Proliferation Activity ◽  
Fine Print

Object. The goal of this study was to evaluate the effect of antisense epidermal growth factor receptor (EGFR) RNA on the growth of rat glioma cells in vitro and in vivo and to determine the feasibility of targeting the EGFR gene for gene therapy in gliomas.Methods. Antisense EGFR complementary (c)DNA was transfected into C6 glioma cells by using lipofectamine. In vitro studies, Southern and Northern blot analyses, in situ hybridization, and immunohistochemical staining were designed to examine the integration and expression of antisense EGFR constructs. The 3′(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay and the average number of argyrophilic nuclear organizer regions (Ag-NORs) were used to evaluate cell proliferation, whereas the terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method and microscopy were used to observe cell apoptosis. As part of the in vivo studies, parental C6 cells and C6 cells transfected with EGFR antisense cDNA were implanted stereotactically into the right caudate nucleus of Wistar rats (C6-injected animals and transfected C6-injected animals). Rats with well-established cerebral C6 glioma foci were treated intratumorally with either antisense EGFR cDNA or empty-vector DNA by using lipofectamine (treated-C6 and control treated group). The general behavior and survival of the rats, findings on magnetic resonance images of their brains, histopathological changes, proliferation activity, and apoptosis of the cerebral gliomas in each group of rats were examined.Exogenous antisense EGFR cDNA was integrated into the genome of C6 cells and expressed. In clones with a high expression of the antisense construct, there was a dramatic decrease in endogenous EGFR messenger RNA and protein levels, reduced proliferation activity, and induction of apoptosis in vitro. The mean survival time of rats injected with C6 cells was 17.3 days. The mean survival time of rats injected with C6 cells followed by treatment with empty vector in lipofectamine was 15.4 days. Survival time was significantly prolonged in 100% of the rats injected with antisense-transfected C6 cells and in two thirds of the rats injected with C6 cells followed by antisense EGFR cDNA. Magnetic resonance imaging revealed distinct cerebral tumor foci in C6-injected rats and in control rats of the treated group, but none were found in the rats injected with transfected C6 cells. Furthermore, tumor foci disappeared completely in C6-injected rats treated with antisense EGFR cDNA. The cerebral gliomas of the rats treated by injection of antisense EGFR RNA were characterized by reduced proliferation activity and the induction of apoptosis.Conclusions. The results of this study indicate that EGFR plays an important role in the genesis of malignant gliomas. It may, therefore, be an effective target of antisense gene therapy in patients with gliomas.

scholarly journals TGF-β1 EXPRESSION BY GLIOMA C6 CELLS IN VITRO

2017 ◽  
Vol 39 (4) ◽  
pp. 258-263 ◽  
Author(s):  
L D Liubich ◽  
L M Kovalevska ◽  
M I Lisyany ◽  
V M Semenova ◽  
T A Malysheva ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Rat Brain ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells ◽  
Fetal Rat ◽  
Tgf Β1 ◽  
Fetal Rat Brain

The aim of the work was to study the impact of fetal rat brain cell supernatant (FRBCS) on the expression of transforming growth factor β1 (TGF-β1) and p53 in C6 cells of rat glioma in vitro. Materials and Methods: FRBCS was obtained from suspensions of fetal rat brain cells on the 14th (E14) day of gestation. C6 glioma cells were cultured for 48 h in the presence of FRBCS or FRBCS + anti-TGF-β1 monoclonal antibody. Immunocytochemical staining for TGF-β1 and p53 was performed. Results: The proportion of TGF-β1-immunopositive tumor cells in C6 glioma cultures was statistically significantly higher than in the control cell cultures of normal fetal rat brain. FRBCS reduced the proportion of TGF-β1-immunopositive tumor cells and increased the proportion of p53-immunopositive cells in C6 glioma cultures. In cells cultured with FRBCS + anti-TGF-β1 monoclonal antibody, the above effects of FRBCS were abrogated. Conclusion: The obtained results suggest that TGF-β1 seems to be responsible for decrease in TGF-β1 expression and increase in p53 expression in C6 glioma cells treated with FRBCS.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

scholarly journals α-Conotoxins and α-Cobratoxin Promote, while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells

Marine Drugs ◽  
2021 ◽  
Vol 19 (2) ◽  
pp. 118
Author(s):  
Tatiana I. Terpinskaya ◽  
Alexey V. Osipov ◽  
Elena V. Kryukova ◽  
Denis S. Kudryavtsev ◽  
Nina V. Kopylova ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Glioma Cells ◽  
Nordihydroguaiaretic Acid ◽  
C6 Glioma ◽  
Cyclooxygenase Inhibitors ◽  
C6 Glioma Cells ◽  
C6 Cells ◽  
Lipoxygenase Inhibitor ◽  
Glioma C6

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, β2 and β4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.

Escitalopram oxalate, a selective serotonin reuptake inhibitor, exhibits cytotoxic and apoptotic effects in glioma C6 cells

2011 ◽  
Vol 23 (4) ◽  
pp. 173-178 ◽  
Author(s):  
Miriş Dikmen ◽  
Zerrin Cantürk ◽  
Yusuf Öztürk
Keyword(s):  
Selective Serotonin Reuptake Inhibitor ◽  
Serotonin Reuptake ◽  
Glioma Cells ◽  
C6 Glioma ◽  
Selective Serotonin ◽  
C6 Glioma Cells ◽  
C6 Cells ◽  
Glioma C6 ◽  
Apoptotic Effects ◽  
Cell Proliferations

Dikmen M, Cantürk Z, Öztürk Y. Escitalopram oxalate, a selective serotonin reuptake inhibitor, exhibits cytotoxic and apoptotic effects in glioma C6 cells.Objective: Various antidepressants, mainly tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), have been reported to exhibit potent anticancer properties in different cancer cells. In this study, we evaluated the antiproliferative and apoptotic effects of escitalopram oxalate (25, 50, 100 and 200 µM) on rat C6 glioma cells.Methods: Cell proliferations were measured by [3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide] (MTT) assay, apoptosis was observed by flow cytometric analysis on C6 cells.Results: Significant decreases in the proliferation of C6 glioma cells were detected depending on increases in the escitalopram concentrations and incubation periods. When compared to controls, C6 cell proliferations after 24 h incubation were determined with 97.7, 85.9, 74.5 and 67.9% for 25, 50, 100 and 200 µM escitalopram, respectively, while the cell proliferations after 48 h were established as 96.5, 68.0, 50.7 and 39.9% for 25, 50, 100 and 200 µM concentrations, respectively. IC50 value of escitalopram was able to be calculated as 106.97 µM after 48 h. Based on Annexin V-propidium iodide (PI) binding capacity for 25, 50, 100 and 200 µM escitalopram, apoptotic effects were determined as 17.0, 22.3, 12.5 and 7.8%, respectively.Conclusion: Based on our findings, escitalopram oxalate was observed to induce cytotoxic and apoptotic activities in C6 cells.

scholarly journals Plasmin modulates the thrombin-evoked calcium response in C6 glioma cells

1994 ◽  
Vol 297 (1) ◽  
pp. 175-179 ◽  
Author(s):  
J S Turner ◽  
G T Redpath ◽  
J E Humphries ◽  
S L Gonias ◽  
S R Vandenberg
Keyword(s):  
Binding Sites ◽  
Cellular Response ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
Similar Response ◽  
C6 Cells ◽  
Plasmin Activity ◽  
Strong Concentration ◽  
Agonist Peptide

Extracellular proteinases may be selectively targeted to cell surfaces by specific receptors or binding sites. In previous studies, we have characterized cellular binding sites for plasminogen and plasmin on rat C6 glioma cells. In this investigation, we studied the response of C6 cells to alpha-thrombin and plasmin by measuring the rapid kinetics of free intracellular Ca2+ concentrations ([Ca2+]i). Thrombin produced a strong, concentration-dependent rise in [Ca2+]i with an onset within 3 s and peak levels achieved in less than 10 s. A similar response was also evoked by an SFLLRN-containing thrombin-agonist peptide. C6 cells did not respond to plasmin (25 nM-1.5 microM). By contrast, pretreatment of C6 cells with 100 nM plasmin significantly inhibited the [Ca2+]i response to thrombin and the thrombin-agonist peptide. The peak [Ca2+]i response to thrombin, in cells pretreated with plasmin, was reduced by approx. 50%. The effect of plasmin on the cellular response to thrombin was selective, as pretreatment of the cells with plasmin did not affect the [Ca2+]i response to platelet-activating factor. Di-isopropylphosphorylplasmin and plasminogen did not inhibit the cellular response to thrombin, indicating that plasmin activity is required and that occupancy of cellular plasmin(ogen)-binding sites alone is insufficient. These studies demonstrate that plasmin does not directly induce a response in C6 cells, but may affect cellular function by specifically modulating the thrombin response.

Bioactivatable, Membrane-Permeant Analogs of Cyclic Nucleotides as Biological Tools for Growth Control of C6 Glioma Cells

2003 ◽  
Vol 384 (9) ◽  
pp. 1321-1326 ◽  
Author(s):  
M. Bartsch ◽  
M. Zorn-Kruppa ◽  
N. Kühl ◽  
H.-G. Genieser ◽  
F. Schwede ◽  
...  
Keyword(s):  
Cyclic Nucleotides ◽  
Growth Control ◽  
Glioma Cells ◽  
Glioma Cell Line ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells ◽  
Dibutyryl Camp ◽  
Ester Prodrugs ◽  
Rat C6 Glioma

Abstract In the present study, the cAMP analogs 8-bromocAMP (8-Brc-AMP), N6-2'O-dibutyryl-cAMP (DBcAMP) and 8-parachlorophenylthio-cAMP (8-CPT-cAMP), as well as the corresponding cAMP-acetoxymethyl (AM)-ester-prodrugs were tested in a HPLC study for their membrane permeability, intracellular accumulation and biotransformation. Antiproliferative activities of these compounds were studied in the rat C6 glioma cell line. Chromatographic analysis revealed that the AM-ester analogs of the cyclic nucleotides penetrate quantitatively into rat C6 glioma cells and generate high amounts of their parent cyclic nucleotides intracellularly within 60 min; however, longterm growth inhibition tested in C6 cells is only slightly enhanced with the AM-ester prodrugs of 8-Br-cAMP or DBcAMP.

Post-transcriptional induction of β1-adrenergic receptor by retinoic acid, but not triiodothyronine, in C6 glioma cells expressing thyroid hormone receptors

1996 ◽  
Vol 135 (6) ◽  
pp. 709-715 ◽  
Author(s):  
Mònica López-Barahona ◽  
Teresa Iglesias ◽  
Irene García-Higuera ◽  
Federico Mayor ◽  
Angel Zaballos ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Thyroid Hormone ◽  
Adrenergic Receptor ◽  
Hormone Receptors ◽  
Glioma Cells ◽  
C6 Glioma ◽  
High Expression ◽  
Thyroid Hormone Receptors ◽  
C6 Glioma Cells ◽  
C6 Cells

López-Barahona M, Iglesias T, García-Higuera I, Mayor Jr F, Zaballos A, Bernal J, Muñoz A. Posttranscriptional induction of β1 -adrenergic receptor by retinoic acid, but not triiodothyronine, in C6 glioma cells expressing thyroid hormone receptors. Eur J Endocrinol 1996:135:709–15. ISSN 0804–4643 Thyroid hormone (triiodothyronine; T3) has been shown to control the expression of β1 -adrenergic receptors (β1-AR) in cardiac myocytes, but not in C6 glioma cells. This cell specificity has been attributed to low expression of T3 receptors and high expression of the c-erbAα2 splice variant that interferes with the action of T3. To check this hypothesis we have expressed the c-erbA/thyroid hormone receptor (TR) α1 gene in C6 glioma cells and investigated their response to thyroid hormone. Cells expressing TRα1, but not wild-type cells, were responsive to T3 as shown by increased expression of mitochrondrial hydroxymethylglutaryl CoA synthase after T3 exposure. However, T3 had no effect on β1-AR gene expression in either set of cells. The β1-AR mRNA concentrations were, however, altered by retinoic acid (RA) treatment. Retinoic acid caused a rapid up-regulation of β1-AR mRNA levels that was blocked by cycloheximide. Retinoic acid did not increase the β1-AR gene transcription rate in run-on experiments. These results indicate an indirect post-transcriptional effect of RA. Control of β1-AR expression in C6 cells is also exerted at the translational level, because there was no correlation between mRNA and protein induction, as determined by radioligand binding studies. We conclude that lack of responsiveness of the β1-AR gene in C6 cells to T3 is not due to high expression of c-erbAα2 but to undefined cell-specific factors. Alberto Muñoz, Instituto Investigaciones Biomedicas, Arturo Duperier 4, 28029 Madrid, Spain

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